Effect of EGFR-induced VEGF on AKT, ERK and p38.

<p>A total of 10⁶ THP1 or Raji cells/well were cultured in the presence of 20ng/ml EGF and the phosphorylation levels of AKT, ERK and p38 were analyzed by Western blot. <b>(A)</b> EGF was treated for the indicated time intervals. Phosphorylation levels of p-AKT, p-ERK and p-p38 increased significantly after EGF stimulation in both THP1 (Left) and Raji (Right) cell lines. <b>(B)</b> In order to measure p-Akt, p-ERK and p-p38, EGFR was blocked using 5μM AG1478 inhibitor for 20min and cells were then stimulated by EGF at 20ng/ml for 10min, 20min, 30min or 5min, 10min, 20min in THP1 or Raji cell lines, respectively. The expression of pAKT, pERK, and p38 was found to be downregulated, using western-blot. It’s worth noting that ERK1/2 and p-ERK1/2 are detected as 2 bands on the western-blot corresponding to ERK1 and ERK2 or their phosphorylated isoforms. Results are representatives of four independent experiments (n = 4), for each time point and treatment condition.</p

EGFR and VEGFR2 are expressed in THP1 and Raji cell lines.

<p>A total of 10⁶ cells/well were cultured. (A) VEGFR1 and VEGFR3 were found to be absent in both cell lines, using RT-PCR. (B) Cells were shown to express EGFR and VEGFR2 mRNA and (C) proteins using RT-PCR and western blot; respectively. Jurkat cell line was used as a negative control whereas monocytes were used as a positive control.</p

Measurement of intracellular calcium after EGF treatment, in the presence or absence of DMH4.

<p>THP1 or Raji cell lines were cultured as 10⁶ cells/well and incubated, or not, with 20<b>μ</b>M DMH4 for lh30min and then treated with 20ng/ml of EGF in a time-course at various time intervals. Fluorometry was then used in order to measure intracellualr calcium concentrations which were significantly and rapidly increased, in both cell lines, but were not affected by VEGFR2 inhibition using 20<b>μ</b>M DMH4. Results are representatives of three independent experiments (n = 3), for each time point and treatment condition, reported as the mean plus or minus the standard error of the mean. *, **, *** indicate p<0.05, p<0.001, p<0.0001; respectively.</p

Effect of EGFR-induced VEGF on phosphorylation of VEGFR2, AKT and ERK.

<p>A total of 10⁶ THP1 or Raji cells/well were cultured in the presence of 20ng/ml EGF. <b>(A)</b> EGF stimulation increased very early the phosphorylation levels of p-VEGFR2 in THP1 (Left) or Raji (Right) cell lines, which peaked at ~5–10 min after treatment. <b>(B)</b> In order to measure p-Akt, p-ERK and p-p38, VEGFR2 was blocked using 20μM DMH4 inhibitor for 1h30min and cells were then stimulated by EGF at 20ng/ml for 10min, 20min, 30min or 5min, 10min, 20min in THP1 or Raji cell lines, respectively. Phosphorylation of AKT and ERK, but not p38, was diminished when the cell lines were pre-incubated with DMH4. <b>(C)</b> Similarly, phosphorylation of AKT and ERK, but not p38, was inhibited when cells were pre-incubated for 1h with 2μg/ml anti-VEGFR2 antibodies. <b>(D)</b> siRNA (20nM) against VEGFR2, left after transfection for 72h, caused a decrease in the phosphorylation levels of AKT and ERK, but not p38. It’s worth noting that ERK1/2 and p-ERK1/2 are detected as 2 bands on the western-blots corresponding to ERK1 and ERK2 or their phosphorylated isoforms. However, when ERK1 antibody is used, only 1 band appears on the blot. Results are representatives of four independent western blot experiments (n = 4).</p

Measurement of ROS production after EGF treatment, in the presence or absence of DMH4 or NAC.

<p>THP1 or Raji cell lines were cultured as 10⁶ cells/well and pretreated for 1h30min with DMH4 (1μM or 20μM) or for 30min with N-acetylcysteine (NAC, 5μM) before treatment with 20ng/ml of EGF in a time-course. Following that, H₂O₂ production was measured using fluorometry. (A) Relative fluorescence curves at the basal level, after stimulation with EGF or after inhibition by DMH4 over a period of time. (B) Quantification of the relative fluorescence curve intensities. ROS production was significantly reduced by VEGFR2 inhibition, using 20μM DMH4. (C) In order to measure p-Akt, p-ERK and p-p38, cells were pretreated with 5μM of the ROS inhibitor N-acetylcysteine (NAC) for 30 min and then treated with EGF at 20ng/ml for 10min, 20min, 30min or 5min, 10min, 20min in THP1 or Raji cell lines, respectively. Western blot analysis showed a significant decrease in pAKT, pERK and p-p38. Therefore, VEGFR2 is responsible for EGF-induced ROS production. Results are representatives of three independent experiments (n = 3), reported as the mean plus or minus the standard error of the mean. *, **, *** indicate p<0.05, p<0.001, p<0.0001; respectively.</p

Bone marrow extracts enhance hematopoietic colony formation.

<p><b>A</b>) About 20,000 CB MNCs from each patient were plated on methylcellulose-based media (HSC002, HSC003, and HSC004), in the presence or absence of BME (CB+BME or CB, respectively). Methylcellulose media contained no growth factors (HSC002), SCF+GM-CSF+IL3+Epo (HSC003), or SCF+GM-CSF+IL3 without Epo (HSC004). Colony forming units (CFU) consisting of CFU-G/M (Granulocyte or Macrophage or both), CFU-E/BFU-E (Erythroid), and CFU-GEMM (Granulocyte Erythroid Macrophage Megakaryocyte) were counted at day 16 using an inverted microscope. Data represents an average of 6 different samples. <b>B</b>) A total of 100 CB CD34+ cells from each patient were plated on methylcellulose media (HSC002, HSC003, and HSC004), in the presence or absence of BME (CB+BME or CB, respectively) as described above. Data represents an average of 4 samples used in the study. Paired TTest was used for statistical significance (*: p<0.05, **: p<0.001).</p

Bone marrow extracts enhance human erythroid engraftment in NSG mice.

<p>Two groups of NSG mice (n = 3 each) received 4×10⁶ CB MNCs previously cultured for 7 days, in the presence or absence of BME. A third group (n = 5) served as a negative control and was injected with saline. Mice were sacrificed three weeks after transplantation. <b>A</b>) The percentage of engraftment was determined using the human pan leukocyte CD45 marker. <b>B</b>) The contribution of myeloid, lymphoid and erythroid populations to total human leukocyte engraftment was determined using CD33, CD19, CD36 and CD45 markers, respectively. *: p<0.05.</p

Bone marrow extracts enhance human bone marrow derived CD34 positive cells engraftment in immunodeficient mice.

<p>Two groups of NSG mice (n = 5 each) received 50×10³ bone marrow derived CD34 positive cells previously cultured for 13 days, in the presence or absence of BME. A third group (n = 5) served as a negative control and was injected with saline. Mice were sacrificed six weeks after transplantation. <b>A</b>) The percentage of engraftment was determined using the human pan leukocyte CD45 marker. <b>B</b>) The contribution of myeloid, lymphoid and erythroid populations to total human leukocyte engraftment was determined using CD33, CD19, CD36 and CD45 markers, respectively. *: p<0.05.</p

Bone marrow extracts enhance human cord blood derived mononuclear cell engraftment in NOD/SCID mice.

<p>Two groups of NOD/SCID mice (n = 5 each) received 3×10⁶ CB MNCs previously cultured for 7 days, in the presence or absence of BME. A third group (n = 5) served as a negative control and were injected with saline. Mice were sacrificed six weeks after transplantation. <b>A</b>) The percentage of engraftment was determined using the human pan leukocyte CD45 marker. <b>B</b>) The contribution of myeloid and lymphoid populations to total human leukocyte engraftment was determined using CD33, CD19 and CD45 markers, respectively. *: p<0.05.</p

Multilineage engraftment of human CB MNC in NSG mice.

<p>In this study, NOD/SCID or NSG mice received <i>iv</i> busulfan conditioning followed by <i>iv</i> injection of CB MNC or CD34⁺ cells that were previously cultured <i>in vitro</i> for 7 days, in the presence or absence of BME. This figure represents NSG mice which received <i>iv</i> injection of 3×10⁶ CB MNC according to the same protocol. Mice were sacrificed and bone marrow cells were harvested from femurs, tibia and pelvis and examined for multilineage engraftment by flow cytometry according to the following gating strategy: <b>A</b>) Live cells were first gated using forward scatter versus side scatter plots (R1 region). The three plots represent mice injected with saline (negative control), CB, or CB+BME; respectively. <b>B</b>) Human leucocytes were then gated using human CD45 staining (pan-leukocyte marker, R2 region). <b>C</b>) From CD45⁺ gate (R2 region), cells were then examined for multi-lineage engraftment defined by the presence of separate lymphoid (CD45⁺CD19⁺, R3 region) and myeloid (CD45⁺CD33⁺, R4 region) populations. <b>D</b>) The population of CD45⁻ cells were gated in order to determine the erythroid populations. Indeed, erythroid populations made up of mature RBCs (CD45⁻CD36⁻CD235a⁺, R5 region) or immature erythroblasts (CD45⁻CD36⁺CD235a⁺, R6 region) were also determined. It’s important to note that the CD36⁺CD235a⁻ population, which is not present in the controls, does not represent immature erythroblasts but are CD45+ mature cells. The percent engraftment was defined as the total number of leucocytes and immature erythroblasts (CD45⁺ and CD45⁻CD36⁺CD235a⁺ cells).</p